Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electric field

ABSTRACT

Methods for reducing peak broadening associated with the establishment of a run field in a capillary electrophoresis process, and apparatus useful for carrying out such methods, are described. Such methods include defining a maximum ramp rate to be used during an initial electric field ramp, defining a minimum period over which the run field is established, and/or maintaining a temperature of a separation medium to within certain ranges during the initial electric field ramp. In addition, methods for determining a ramp rate effective for reducing peak broadening are disclosed.

FIELD OF THE INVENTION

This invention relates to methods and apparatus for performing capillaryelectrophoresis. More specifically, this invention is directed towardsmethods and apparatus useful for reducing peak broadening caused duringthe establishment of a run field used to conduct the capillaryelectrophoresis process.

BACKGROUND

Electrophoretic separations of biopolymers and small molecules arecritically important in modern biology and biotechnology, playing acentral role in such techniques as DNA sequencing, protein molecularweight determination, genetic mapping, and the like. A particularlypreferred electrophoresis format is capillary electrophoresis (CE),where the electrophoresis is performed in a capillary channel having asmall internal diameter, e.g., between 5 and 100 μm. In manyapplications, capillary electrophoresis results in enhanced separationperformance over traditional slab-based formats because the superiorability of the narrow-bore capillary to dissipate Joule heat allows forthe use of high electrical fields thereby resulting in fast separationsin which the effect of analyte diffusion is reduced. In addition,capillary electrophoresis is well adapted to automation because of theability to automate the steps of sample loading, analyte detection, andreplenishment of the separation medium.

Certain commercially important applications of capillary electrophoresisrequire exquisite separation efficiency. For example, in DNA sequencingseparations, plate counts of 20 million plates per meter may berequired. In order to achieve this kind of performance, everythingpossible must be done to reduce instrumental effects that can lead topeak broadening and therefore lower separation efficiencies, e.g., peakbroadening caused by the radial temperature profile within the lumen ofthe capillary, the sample injection volume, solute-wall interactions,siphoning, finite detection volume, and the like (e.g., CapillaryElectrophoresis Theory and Practice, Chap. 1, Grossman and Colburn,eds., Academic Press (1992)). In addition, because of the highthroughput requirements of large-scale DNA sequencing operations, anymeasures taken to increase the separation performance of theelectrophoretic analysis preferably will not substantially reduce thespeed, and therefore the throughput, of the process.

Therefore, any further understanding of the mechanisms underlying peakbroadening and techniques for reducing the impact of such mechanisms onthe performance of CE separations without sacrificing the speed ofanalysis would be an important contribution to the field of capillaryelectrophoresis and related applications.

SUMMARY

The present invention is directed towards the discovery of methods andapparatus useful for increasing the separation performance of capillaryelectrophoresis separations performed in a fluid separation medium bycontrolling the rate of increase of the electric field strength and/orthe temperature of the separation medium during aninitial-electric-field-ramp stage of the capillary electrophoresisprocess.

In one aspect, the invention comprises a capillary electrophoresismethod wherein a run field is established during an initial electricfield ramp in a controlled manner according to a pre-defined ramp rate.In a preferred embodiment of this aspect of the invention, the run fieldis established at a ramp rate no greater than about 5 V/cm-s. In anotherpreferred embodiment of this aspect of the invention, the run field isestablished over a period of at least about ten seconds. In yet anotherpreferred embodiment of this aspect of the invention, the run field isestablished at a ramp rate which results in a reduction in the amount ofpeak broadening associated with the establishment of the run field by atleast about 10%. In another preferred embodiment of this aspect of theinvention, the run field is established at a ramp rate which results inan increase in a length of read of at least about 20 nucleotides overthat achieved when the run field is not established in a controlledmanner.

In another aspect, the invention comprises a method for producing adesired reduction in an amount of peak broadening caused duringestablishment of a run field comprising, for each of a plurality ofelectrophoretic runs, establishing the run field at each of a pluralityof different ramp rates, at least some of which ramp rates are notgreater than about 5 V/cm-s; analyzing a degree of peak broadeningobserved for each run; and selecting a ramp rate which is no greaterthan that which produced a desired reduction in peak broadening.

In yet another aspect, the present invention comprises a capillaryelectrophoresis method in which analyte species are separated bydifferential electrophoretic migration through a fluid separation mediumlocated within a capillary under the influence of a run field, animprovement for reducing the peak broadening associated with theestablishment of the run field comprising reducing a temperature of anenvironment surrounding the capillary during an initial electric fieldramp. In one preferred embodiment, the temperature of the environmentsurrounding the capillary is reduced by an amount sufficient to maintainan average temperature of the separation medium during such initialelectric field ramp to within about 0.4° C. of the average temperatureof the separation medium prior to initiating the initial electric fieldramp. In another preferred embodiment, the temperature of theenvironment surrounding the capillary is reduced by an amount sufficientto maintain an average temperature of the separation medium during suchinitial electric field ramp substantially constant with respect to anaverage temperature of the separation medium prior to initiating theinitial electric field ramp to an extent sufficient to result in adisplacement of the fluid separation medium at an inlet end of thecapillary during the initial electric field ramp of less than about 600μm. In yet another preferred embodiment, the temperature of theenvironment surrounding the capillary is reduced by an amount sufficientto maintain an average temperature of the separation medium during suchinitial electric field ramp substantially constant with respect to anaverage temperature of the separation medium prior to initiating theinitial electric field ramp to an extent sufficient to increase a lengthof read by at least about 20 nucleotides.

These and other aspects, embodiments and features of the presentinvention will become better understood with reference to the followingdescription, drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C and 1D show schematic depictions of several exemplaryinitial electric field ramps comprising various alternativediscontinuous multi-step electric field vs. time profiles.

FIG. 2 shows several schematic depictions of exemplary initial electricfield ramps comprising various alternative continuous electric field vs.time profiles.

FIG. 3 shows a plot of LOR vs. migration time of a 700-nucleotide DNAfragment in approximately 96 different capillaries at each of a numberof run fields without the use of an initial electric field ramp andusing a separation medium containing 8M urea.

FIG. 4 shows a plot of LOR vs. migration time of a 700-nucleotide DNAfragment in approximately 96 different capillaries at each of a numberof run fields without the use of an initial electric field ramp andusing a separation medium containing 6M urea.

FIG. 5 shows a plot of LOR vs. migration time of a 700-nucleotide DNAfragment in approximately 96 different capillaries at each of a numberof run fields with the use of an initial field ramp of 0.25 V/cm-s andusing a separation medium containing 8M urea.

FIG. 6 shows a plot of LOR vs. migration time of a 700-nucleotide DNAfragment in approximately 96 different capillaries at each of a numberof run fields with the use of an initial field ramp of 0.25 V/cm-s andusing a separation medium containing 6M urea.

FIG. 7 shows the effect of various ramp rates of an initial electricfield ramp on LOR using a run field of 160 V/cm and a separation mediumcontaining 8M urea.

FIG. 8 shows the effect of various ramp rates of an initial electricfield ramp on the limit of resolution using a run field of 120 V/cm anda separation medium containing 6M urea.

FIG. 9 shows the effect of using an initial electric field ramp having aramp rate of 0.25 V/cm-s on LOR as a function of run field in aseparation medium containing 8M urea.

FIG. 10 shows the effect of using an initial electric field ramp havinga ramp rate of 0.25 V/cm-s on LOR as a function of run field in aseparation medium containing 6M urea.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that such embodiments arenot intended to limit the invention to those embodiments. On thecontrary, the invention is intended to cover alternatives,modifications, and equivalents, which may be included within the scopeof invention as deliniated by the appended claims.

The invention is based in part on the discovery that a substantialamount of peak broadening is caused when a run field is firstestablished, and that such peak broadening can be greatly reduced byestablishing the run field in a controlled manner during an initialelectric field ramp, and/or by maintaining the temperature of theseparation medium at a constant value during the establishment of therun field.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Separation medium” means a medium typically located within the lumen ofa capillary through which an electrophoretic separation is conducted.Exemplary separation media include crosslinked gels, un-crosslinkedpolymer solutions, or polymer-free solvents, e.g., buffered water.Optionally, separation media may include denaturants such as detergents,e.g., SDS, or organics, e.g., urea or formamide.

“Fluid separation medium” means an electrophoretic separation mediumthat cannot sustain a shearing stress in the absence of motion, i.e., amedium that flows in response to a shearing stress. Examples of fluidseparation medium include but are not limited to liquids and liquidsolutions, e.g., buffered aqueous polymer solutions.

“Run field” means an electric field used to conduct an electrophoresisseparation. Typical run fields used in capillary electrophoresis rangefrom about 50 V/cm to about 3000 V/cm or greater.

“Initial electric field ramp” means an electric field versus timeprofile during a period in which a run field is initially established.

“Ramp rate” means a run field, E, divided by a total elapsed time of aninitial electric field ramp, T. For example, if a run field is 100 V/cmand an initial field ramp takes ten seconds to reach the run field, thanthe ramp rate is 10 V/cm-s.

“Capillary” or “capillary tube” means tubes or channels or otherstructure capable of supporting a volume of separation medium. Thegeometry of a capillary may vary widely and includes tubes withcircular, rectangular or square cross-sections, channels, groved plates,and the like. Capillaries may be fabricated by a wide range of wellknown technologies, e.g., pulling, etching, photolithography, and thelike. An important feature of a capillary for use with the invention isthe surface-area-to-volume ratio of the capillary lumen. High values ofthis ratio permit efficient dissipation of Joule heat produced duringelectrophoresis. Preferably, ratios in the range of about 0.4 to 0.04μm⁻¹ are employed. These ratios correspond to surface-to-volume ratiosof tubular capillaries with circular cross-sections having insidediameters in the range of about 10 μm to about 100 μm. Capillaries maybe formed as individual elements, or as channels formed in a monolithicsubstrate (e.g., Pace, U.S. Pat. No. 4,908,112; Soane and Soane, U.S.Pat. No. 5,126,022). Capillaries include an “inlet end” through whichsample analytes are introduced into the lumen of the capillary.

II. Description of the Preferred Embodiments

When a run field is established in a CE capillary, the temperature of aseparation medium located in a lumen of the capillary will generallyincrease due to Joule heating (e.g., Capillary Electrophoresis Theoryand Practice, Chap. 1, Grossman and Colburn, eds., Academic Press,(1992)). Joule heating will result in both an increase in the averagetemperature of the separation medium, ΔT, and the formation of aparabolic radial temperature profile within the separation medium suchthat the temperature of the separation medium is higher at thecenterline of the lumen and lower at the periphery.

The magnitude of the temperature rise ΔT is a function of a number ofparameters including but not limited to the shape, materials anddimensions of the capillary, the thermal conductivity of the separationmedium and capillary materials, the efficiency of heat transfer betweenthe capillary and the surrounding environment, the electricalconductivity of the separation medium, and the magnitude of thetemperature dependence of the electrical conductivity of the separationmedium. The magnitude of ΔT is generally a non-linear function of theseparameters.

Generally, when a material is heated, because of the more intensethermal vibration of its constituent atoms, the material expands. Theextent to which a material expands in response to a temperature increaseis characterized in terms of its thermal expansion coefficient, where,as used herein, the term “thermal expansion coefficient” means aproportionality constant relating an increase in a dimension of amaterial, e.g., length, area or volume, to an original dimension of thematerial, as the result of an increase in the temperature of thematerial. For example, to a first approximation, the increase in thelength of a material due to thermal expansion, ΔL/L, is proportional tothe change in temperature, ΔT, according to the relation

ΔL/L=α _(L) ΔT

where α_(L) is the linear expansion coefficient of the material.However, closer examination shows that α_(L) is generally not absolutelyconstant, but rather increases slightly with temperature. Exemplaryvalues of the linear expansion coefficient of several selected materialsare given in Table 1 immediately below.

TABLE 1 Exemplary Thermal Expansion Coefficients Linear ThermalExpansion Coefficient Material x 10⁶/° C. (α_(L)) Fused Silica 0.5Borosilicate Glass* 3 Polyvinylchloride* 54 Polyethylene* 180 DI Water**360 POP-6 Polymer** 580 *Perry's Chemical Engineer's Handbook, SixthEdition, page 9-98, Table 6-43, McGraw Hill, (1984). **Experimentallymeasured value.

When a fluid material is subject to thermal expansion in a channelhaving rigid walls and having at least one open end, a flow will beinduced, resulting in a finite displacement of the fluid material. Thepresent inventors have discovered that such expansion-driven flows are,under certain operating conditions, present during an initial electricfield ramp portion of a capillary electrophoresis process, and thatdisplacements of the separation medium caused by such flows cannegatively impact separation performance. Moreover, the inventors havedetermined this effect has been found to be particularly pronounced insystems similar to those frequently encountered in the CE art, i.e.,systems employing a fluid separation medium, a high electric field, anda long narrow channel formed from a rigid material, e.g., a fused-silicacapillary tube having an internal diameter of 50 μm and a length of 50cm. In particular, to a first approximation, in terms of the extent ofdisplacement of the fluid separation medium during an initial electricfield ramp, the present inventors have discovered that the displacementwill (1) increase with increasing capillary length, (2) will increasewith increasing thermal expansion coefficient of the fluid separationmedium, (3) will increase with increasing electrical conductivity of thefluid separation medium, (4) will increase with decreasing heat transfercoefficient between the exterior surface of the capillary and thesurroundings, (5) will increase with increasing run field, (6) and willincrease with increasing capillary internal diameter.

While the foregoing theoretical discussion has been provided to aide inthe understanding and description of the present invention, thediscussion should in no way be deemed to limit the scope of theinvention to this or any other particular theoretical formulation.

In a first aspect, the present invention comprises methods and apparatusfor reducing the amount of peak broadening caused by the uncontrolledestablishment of a run field in a CE separation employing a fluidseparation medium. More specifically, this aspect of the inventioncomprises a capillary electrophoresis system in which analyte species,e.g., nucleic acids, are separated by differential electrophoreticmigration through a fluid separation medium under the influence of a runfield E, wherein the run field is established during an initial electricfield ramp in a controlled manner according to a defined electric-fieldvs. time profile having a characteristic ramp rate.

Exemplary preferred electric field vs. time profiles according to thepresent invention include but are not limited to discontinuousmulti-step profiles, continuous linear profiles, continuous non-linearprofiles, or combinations of such discontinuous and continuous profiles.In addition, the elapsed time T of the initial electric field ramp andthe value of the run field E may vary as required by a particularapplication. Preferably, the initial electric field ramp will comprise afield vs. time profile that serves to both reduce peak broadening and toachieve the run field E in as short an elapsed time T as possibleconsistent with the desired reduction in peak broadening.

In one preferred embodiment, the initial electric field ramp ischaracterized by a field vs. time profile comprising a plurality ofdiscontinuous steps where each step is characterized by a particularchange in electric field, ΔE, and time, Δt. FIG. 1A shows a schematicdepiction of such a discontinuous multi-step field vs. time profile.Generally, as ΔE becomes smaller and/or Δt becomes larger, the impact ofthe initial electric field ramp on peak broadening is reduced.

In one particularly preferred embodiment, the initial electric fieldramp is characterized by an electric field vs. time profile comprising aplurality of discontinuous steps wherein the values of ΔE and Δt foreach step are equal, e.g., as schematically depicted in FIG. 1A. Thus,to characterize such an electric field vs. time profile, one need onlyspecify a value for the run field E, the change in electric field ateach step ΔE, and the time for each step Δt.

In another preferred embodiment, the initial electric field ramp ischaracterized by an electric field vs. time profile comprising aplurality of discontinuous steps wherein the values of ΔE and Δt foreach step are not equal, e.g., as schematically depicted in FIGS. 1B, 1Cand 1D. FIG. 1B shows a schematic depiction of an initial electric fieldramp comprising a discontinuous multi-step field vs. time profile inwhich the values of both ΔE and Δt increase at each step. Alternatively,FIG. 1C shows a schematic depiction of an initial electric field rampcomprising an electric field vs. time profile in which the value of ΔEdecreases at each step and the value of ΔT is the same for each step.Finally, FIG. 1D shows a schematic depiction of an initial electricfield ramp comprising an electric field vs. time profile in which thevalues of both ΔE and Δt vary chaotically at each step. Because thetemperature rise inside the capillary lumen caused by Joule heating istypically an increasing non-linear function of the electrical fieldstrength, the field vs. time profile depicted schematically in FIG. 1Cin which the value of ΔE decreases at each step is particularlypreferred because it both serves to reduce the impact of the initialfield ramp on peak broadening while at the same time reducing theelapsed time T of the initial electric field ramp.

While the values of E, ΔE and Δt will vary depending upon therequirements of a particular application, preferably, when employing aninitial electric field ramp comprising a discontinuous multi-stepelectric field vs. time profile, the value of the run field E is betweenabout 50 V/cm and 3000 V/cm, the time per step Δt is between about 5 sand 200 s, and the change in electric field per step ΔE is between about1 V/cm and 200 V/cm. More preferably E is between about 80 V/cm and 320V/cm, Δt is between about 10 s and 100 s, and ΔE is between about 2 V/cmand 60 V/cm.

In another preferred embodiment, the initial electric field ramp ischaracterized by a continuous electric field vs. time profile. As shownin FIG. 2, the continuous electric field vs. time profile may be linear,(e.g., curve 1 in FIG. 2), concave (e.g., curve 2 in FIG. 2), convex(e.g., curve 3 in FIG. 2), or various combinations of linear, concave,and/or convex (e.g., curve 4 in FIG. 2). As discussed with respect toFIG. 1C, preferably the continuous field vs. time profile is linear orconvex due to the usual non-linearity of the temperature rise caused byJoule heating.

In another preferred embodiment of this first aspect of the invention,the initial electric field ramp is characterized by a field vs. timeprofile comprising a combination of a stepped and a continuous profile.

The particular range of values of the ramp rate of the initial electricfield ramp that will result in reduced peak broadening and thereforeimproved separation performance will depend on a number of experimentalparameters including the shape, materials and dimensions of thecapillary, particularly the length of the capillary, the thermalconductivity of the separation medium and the capillary, the differencein the thermal expansion coefficient of the separation medium and thecapillary, the efficiency of heat transfer between the capillary and thesurrounding environment, the electrical conductivity of the separationmedium, and the temperature dependence of the electrical conductivity ofthe separation medium. In particular, to a first approximation, asmaller ramp rate will be required with (1) increasing capillary length,(2) increasing thermal expansion coefficient of the fluid separationmedium, (3) increasing electrical conductivity of the fluid separationmedium, (4) decreasing heat transfer coefficient between the exteriorsurface of the capillary and the surroundings, (5) increasing run field,and (6) increasing capillary internal diameter.

Therefore, to determine a ramp rate of an initial electric field rampthat will result in reduced peak broadening under a given set ofoperational conditions, the following procedure may be used. First, foreach of a plurality of electrophoretic runs, establish a run field usingan initial electric field ramp, where each run uses a different ramprate, and where at least some of the ramp rates are less than or equalto about five V/cm-s. Next, analyze the extent of peak broadeningobserved for each run, e.g., by measuring a peak width at half height ofa selected peak in each run. Finally, select as the preferred ramp rateone that results in a desired degree of reduction in peak broadening.

Regardless of the particular ramp profile used, e.g., step, continuous,or a combination of stepped and continuous, preferably the ramp rate ofthe initial electric field ramp is less than about five V/cm-s. Morepreferably, the ramp rate of the initial electric field ramp ranges fromabout 0.05 V/cm-s to about 3.0 V/cm-s. In a particularly preferredembodiment, the ramp rate ranges from about 0.1 V/cm-s to about 1.0V/cm-s.

Alternatively, the run field should be established using an initialelectric field ramp having an elapsed time T of at least about tenseconds. More preferably, the run field should be established using aninitial electric field ramp having an elapsed time ranging from about 20seconds to about 500 seconds. More preferably, the run field should beestablished using an initial electric field ramp having an elapsed timeranging from about 500 seconds to about 4000 seconds.

Preferably, the run field E ranges between about 50 and 3000 V/cm, morepreferably between about 80 and 500 V/cm.

In yet another preferred embodiment of this first aspect of the presentinvention, the run field is established using an initial electric fieldprofile having a ramp rate which results in a reduction in an amount ofpeak broadening associated with the establishment of the run field of atleast about 10% as compared to that found when an initial electric fieldramp is not used, and more preferably a reduction in an amount of peakbroadening of at least about 25%, and even more preferably a reductionin an amount of peak broadening of at least about 40%.

In another preferred embodiment of the present invention, the analytespecies is nucleic acid and the run field is established using aninitial electric field profile having a ramp rate which results in anincrease in a length of read of at least about 20 nucleotides,preferably about 40 nucleotides, and more preferably about 80nucleotides, as compared to that found when an initial electric fieldramp is not used.

In another aspect, the present invention comprises methods and apparatusfor reducing the amount of peak broadening caused by the application ofa run field in a CE separation by reducing a temperature of anenvironment surrounding the capillary during an initial electric fieldramp by an amount sufficient to maintain the average temperature of theseparation medium at a substantially constant value during such initialelectric field-ramp. As used herein, the term “average temperature”refers to a spatial average temperature where the temperature isaveraged across a dimension normal to the direction of electrophoreticmigration, e.g., in the case of a cylindrical capillary, the dimensionis the radius.

In particular, according to the method of this aspect of the presentinvention, as the electric field across the capillary is increasedduring the initial electrical field ramp, the temperature of theenvironment surrounding the capillary is reduced by an amount effectiveto maintain the average temperature of the separation medium locatedwithin the lumen of the capillary within about 0.4° C. of the averagetemperature of the separation medium prior to initiating the initialelectric field ramp, more preferably to within about 0.2° C. of theaverage temperature of the separation medium prior to initiating theinitial electric field ramp, and even more preferably to within about0.1° C. of the average temperature of the separation medium prior toinitiating the initial electric field ramp.

In another preferred embodiment of this second aspect of the presentinvention, during the initial electric field ramp, the temperature ofthe fluid separation medium located within the lumen of the capillary ismaintained constant with respect to the average temperature of theseparation medium prior to initiating the initial electric field ramp byreducing the temperature of the environment surrounding the capillary byan amount sufficient to result in a displacement of the fluid separationmedium at an inlet end of the capillary during the initial electricfield ramp of less than about 600 μm, and preferably less than about 200μm, and more preferably less than about 20 μm.

In one preferred embodiment of this aspect of the invention, thetemperature of the separation medium located within the lumen of thecapillary is monitored by measuring the relationship between theelectric field applied across the capillary and the measured currentpassing through the capillary. By knowing how the conductivity of aparticular separation medium varies as a function of temperature, it ispossible to obtain an accurate measure of the temperature of theseparation medium as the electric field is increased. Thus, as thetemperature of the separation medium begins to rise, the temperature ofthe surroundings is lowered using active feedback control.Alternatively, the temperature of the separation medium located withinthe lumen of the capillary is maintained substantially constant using apre-programmed temperature ramp based on prior knowledge of the medium'stemperature vs. electric field characteristics.

For the most part, with the exception of the improvements embodied inthe present invention, the methods and apparatus used to carry out theCE separations according to the present invention may be performed usingconventional CE methods and apparatus, as generally described elsewhere(e.g., Capillary Electrophoresis Theory and Practice, Grossman andColburn, eds., Academic Press (1992)). For example, standard capillarytubes, e.g., polyimide-coated fused silica capillary tubes, fluidseparation medium, e.g., buffered polymer solutions or polymer freebuffer solutions, sample injection techniques, e.g., electrokinetic orhydrodynamic injection, automated system control devices, e.g., adigital computer, and detection techniques, e.g., fluorescence oroptical absorbence, may utilized to practice the methods of theinvention.

However, CE systems used to carry out the methods of the presentinvention include certain non-standard features and capabilities. Thepower supply portion of a CE system for use in the present inventionshould be controllable by a programmable electronic controller, e.g., apersonal computer, so as to allow for the control of an electric fieldvs. time profile of an initial electric field ramp. In addition, whereactive feedback control of the initial electric field ramp is used, theelectronic controller should be connected to a current monitor formonitoring the electric current passing through the electrophoresiscapillary and be able to automatically adjust the electric field acrossthe capillary in response to the current measurement. Also, the CEsystem for use with the present invention should employ a temperaturecontrol system for controlling the temperature of the environmentsurrounding the capillary channel. The temperature control system shouldbe capable of controlling the temperature of the surrounding environmentto within about 0.1° C. along substantially the entire length of thecapillary, be capable of changing the temperature of the surroundingenvironment in increments of 0.1° C., and be capable of adjusting thetemperature of the surrounding environment during an initial electricfield ramp, where, optionally, such changes are in response to changesin the temperature of the separation medium located in the capillarylumen.

III. EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

Materials and Methods

All electrophoretic separations were performed using an ABI PRISM™ 3700DNA Analyzer (PE Biosystems, p/n 4308058) equipped with a 50 cmcapillary array (p/n 4305787). The 3700 system includes approximately 96separate fused-silica separation capillaries, each capillary having anuncoated interior surface, a total length of 50 cm, an effectiveseparation length of 50 cm, and in internal diameter of 50 μm.Fluorescence detection of the sample analytes in the 3700 system isaccomplished using a sheath-flow detection system (e.g., Kambara et al.,U.S. Pat. No. 5,529,679; Dovichi et al., U.S. Pat. No. 5,439,578).

A standard sample mixture comprising 20 single stranded DNA fragmentshaving sizes of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350,400, 450, 490, 500, 550, 600, 650, and 700 nucleotides was used tocharacterize separation performance under various operating conditions.Each fragment of the mixture was labeled with TET dye. The samplemixture was dissolved in deionized formamide and 0.3 mM disodium EDTA toa final concentration of about 0.03 nM.

The temperature of the capillary array was maintained at 50° C.±0.1° C.Samples were electrokinetically injected into the capillaries byapplying an electric field of 50 V/cm for 30 s while the inlet ends ofthe capillaries were immersed in the sample mixture.

The separation medium used was a modified version of the commerciallyavailable ABI PRISM™ 3700 POP6 polymer (PE Biosystems, p/n 4306733) inwhich the denaturant was replaced with an alternative denaturantcomprising urea at a concentration of either 6M or 8M. The POP6 polymeris a solution of a linear substituted polyacylamide.

Each electropherogram was characterized using a parameter called the“limit of resolution,” or “LOR.” The LOR is defined as a point in anelectropherogram, expressed in terms of the size of a polynucleotidefragment located at that location in the electropherogram, at which thefollowing relation is satisfied,$0.5 = \frac{X_{2} - X_{1}}{\frac{1}{2}\left( {W_{1} + W_{2}} \right)}$

where X₂ is the location of the center of a peak 2, X₁ is the locationof the center of a peak 1, W₁ is the peak width at half height of peak1, and W₂ is the peak width at half height of peak 2, where peaks 1 and2 are DNA fragments that differ in size by a single nucleotide. Becausethe actual fragments in the standard mixture differ in size by more thana single nucleotide, the single-nucleotide interval (X₂−X₁) wasestimated by plotting the position X of each of the 20 fragments in thesample mixture as a function of size, fitting the data points with athird-order polynomial, and determining a single-nucleotide calculatedinterval (X₂−X₁) based on an interpolation of the resulting fittedcurve. The peak widths at half height, W₁ and W₂, were estimated byassuming that the peak widths for fragments differing in size by asingle nucleotide were the same as those of the nearest peaks of the 20actual peaks. In practice, actual automated base-calling algorithms cantypically call bases 100 to 200 nucleotides past the LOR, thus, if for agiven separation the LOR was 500, one could expect accurate (i.e.,greater than about 98% accuracy) base calling out to about 700nucleotides.

Example 1 Effect of Run Voltage on the LOR in Two Different SeparationMedia

FIGS. 3 and 4 show, at each of a number of different run fields, the LORvalue for each of about 96 different electropherograms. In FIG. 3 theseparation medium contained 8 M urea denaturant while in FIG. 4 theseparation medium contained 6 M urea. The y-axis of each plot indicatesthe LOR value, while the x-axis of each plot indicates the time requiredfor a 700 nucleotide fragment to reach the detector, T₇₀₀. As can beclearly seen from the data, for both separation media, as the electricfield is increased, and thus T₇₀₀ decreased, the average value of theLOR, LOR_(avg), decreased, and the standard deviation of the LOR,SD_(LOR), increased. In addition, the magnitude of SD_(LOR) was large incomparison with LOR_(avg). Values for LOR_(avg) and SD_(LOR) for thedata of FIGS. 3 and 4 are presented in Tables 2 and 3 immediately below.Thus, these data indicate that when the speed of the analysis isincreased by increasing the run field, a substantial penalty of reducedLOR and increased SD_(LOR) is paid. For example, according to the datapresented in Table 2 below, by increasing the run field from 90 V/cm to130 V/cm, a reduction in LOR of 165 nucleotides was observed.

TABLE 2 FIG. 3 Data (8 M Urea) E (V/cm) LOR_(avg) SD_(LOR) 90 632 29 105583 48 120 528 58 130 467 69

TABLE 3 FIG. 4 Data (6 M Urea) E (V/cm) LOR_(avg) SD_(LOR) 80 501 93 105461 53 120 407 93

Example 2 Effect of An Initial Electric Field Ramp on LOR as a Functionof Run Field

FIGS. 5 and 6, when compared with FIGS. 3 and 4, show the effect of acontrolled initial electric field ramp on the LOR as a function of runfield in two different separation media. In FIG. 5 the separation mediumcontained 8 M urea denaturant while in FIG. 6 the separation mediumcontained 6 M urea. In both the experiments of FIG. 5 and FIG. 6, theinitial electric field ramp was 0.25 V/cm-s using a discontinuous stepelectric field vs. time profile where ΔE was 10 V/cm and Δt was 40 s ateach step. As can be clearly from the data, the introduction of theinitial electric field ramp resulted in a substantial increase inLOR_(avg) and decrease in SD_(LOR) at each of the run fields studied.However, the effect was most pronounced for the higher electric fields.Thus, these data indicate that by using a controlled initial electricfield ramp according to the present invention, higher run fields can beused without sacrificing separation performance, and consequentlyconsiderably shorter analysis times may be achieved, leading tosignificantly higher overall throughput. Values for LOR_(avg) andSD_(LOR) for the data of FIGS. 5 and 6 are presented in Tables 4 and 5immediately below.

TABLE 4 FIG. 5 Data (8 M Urea) E (V/cm) LOR_(avg) SD_(LOR) 90 611 29 105621 41 120 619 40 140 608 49 150 589 32

TABLE 5 FIG. 6 Data (6 M Urea) E (V/cm) LOR_(avg) SD_(LOR) 80 643 33 100645 24 120 643 33

Example 3 Effect of Ramp Rate on LOR

FIGS. 7 and 8 show the effect of the ramp rate of the initial electricfield ramp on LOR at two different run fields in two differentseparation media. Each of the initial electric field ramps comprised adiscontinuous step electric field vs. time profile in which for eachramp rate the values of ΔE and Δt were equal.

In FIG. 7 the run field was 160 V/cm and the separation medium contained8 M urea. The different ramp rates shown in FIG. 7 were achieved byholding ΔE at 500 V and varying Δt between 10 and 80 seconds. As can beclearly seen by the data in FIG. 7, using a ramp rate of about 0.12V/cm-s resulted in an increase of LOR of almost 80 nucleotides.

In FIG. 8 the run field was 120 V/cm and the separation medium contained6 M urea. The different ramp rates were achieved by holding ΔE at 500 Vand varying Δt between 5 and 40 seconds. As can be clearly seen by theplot in FIG. 8, using a ramp rate of about 0.25 V/cm-s resulted in anincrease of LOR of almost 100 nucleotides.

Example 4 Effect of Using an Initial Electric Field Ramp on LOR as aFunction of Run Field

FIGS. 9 and 10 demonstrate the effect of using an initial electric fieldramp on LOR as a function of run field. The ramp rate of the initialelectric field ramp was 0.25 V/cm-s in both cases where the initialelectric field ramps were achieved using a discontinuous step electricfield vs. time profile in which the value of ΔE was 10 V/cm and thevalue of Δt was 40 s. In FIG. 9, the separation medium contained 8M ureaand in FIG. 10 the separation medium contained 6M urea. The plots shownin both FIGS. 9 and 10 clearly illustrate the dramatic effect that aninitial electric field ramp can have on separation performance, and alsoindicate that this effect becomes more pronounced at higher run fields.The dramatic effect of using an initial electric field ramp on the speedof a separation can also bee clearly seen in the data of FIGS. 9 and 10.For example, referring to FIG. 10, to achieve a LOR of 600 without theuse of an initial electric field ramp, the run field cannot exceed 100V/cm, while, if an initial electric field ramp of 0.25 V/cm-s isemployed, a run field of 150 V/cm may be used, resulting in a decreasein analysis time of approximately 50% (not including the ten minutesrequired to effect the initial electric field ramp).

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the electrophoresis art will clearlyunderstand that many modifications are possible in the preferredembodiment without departing from the teachings thereof. For example,one may practice the present invention using both controlled initialelectric field ramp and temperature ramp protocols in combination. Allsuch modifications are intended to be encompassed within the followingclaims.

We claim:
 1. In a capillary electrophoresis method in which analytespecies are separated by differential electrophoretic migration througha fluid separation medium located within a capillary under the influenceof a run field, an improvement for reducing peak broadening associatedwith the establishment of the run field comprising: reducing atemperature of an environment surrounding the capillary during aninitial electric field ramp by an amount sufficient to maintain anaverage temperature of the separation medium during such initialelectric field ramp to within about 0.4° C. of the temperature of theseparation medium prior to initiating the initial electric field ramp.2. The method of claim 1 wherein the temperature of the separationmedium during an initial electric field ramp is maintained to withinabout 0.2° C. of the temperature of the separation medium prior toinitiating the initial electric field ramp.
 3. The method of claim 1wherein the temperature of the separation medium during an initialelectric field ramp is maintained to within about 0.1° C. of thetemperature of the separation medium prior to initiating the initialelectric field ramp.
 4. In a capillary electrophoresis method in whichanalyte species are separated by differential electrophoretic migrationthrough a fluid separation medium located within a capillary under theinfluence of a run field, an improvement for reducing peak broadeningassociated with the establishment of the run field comprising: reducinga temperature of an environment surrounding the capillary during aninitial electric field ramp by an amount sufficient to maintain anaverage temperature of the separation medium during such initialelectric field ramp substantially constant with respect to an averagetemperature of the separation medium prior to initiating the initialelectric field ramp to an extent sufficient to result in a displacementof the fluid separation medium at an inlet end of the capillary duringthe initial electric field ramp of less than about 600 μm.
 5. The methodof claim 4 wherein the displacement of the fluid separation mediumduring the initial electric field ramp of less than about 200 μm.
 6. Themethod of claim 4 wherein the displacement of the fluid separationmedium during the initial electric field ramp of less than about 20 μm.